Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2012, Article ID 324832, 8 pagesdoi:10.1155/2012/324832

Review Article

The Human Tripeptide GHK-Cu in Prevention ofOxidative Stress and Degenerative Conditions of Aging:Implications for Cognitive Health

Loren Pickart, Jessica Michelle Vasquez-Soltero, and Anna Margolina

Skin Biology, Research & Development Department, 4122 Factoria Boulevard SE-Suite No. 200, Bellevue, WA 98006, USA

Correspondence should be addressed to Loren Pickart, lorenpickart@skinbiology.com

Received 13 January 2012; Accepted 27 February 2012

Academic Editor: Marcos Dias Pereira

Copyright © 2012 Loren Pickart et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Oxidative stress, disrupted copper homeostasis, and neuroinflammation due to overproduction of proinflammatory cytokines areconsidered leading causative factors in development of age-associated neurodegenerative conditions. Recently, a new mechanismof aging—detrimental epigenetic modifications—has emerged. Thus, compounds that possess antioxidant, anti-inflammatoryactivity as well as compounds capable of restoring copper balance and proper gene functioning may be able to prevent age-associated cognitive decline and ward off many common neurodegenerative conditions. The aim of this paper is to bring attentionto a compound with a long history of safe use in wound healing and antiaging skin care. The human tripeptide GHK was discoveredin 1973 as an activity in human albumin that caused old human liver tissue to synthesize proteins like younger tissue. It has highaffinity for copper ions and easily forms a copper complex or GHK-Cu. In addition, GHK possesses a plethora of other regenerativeand protective actions including antioxidant, anti-inflammatory, and wound healing properties. Recent studies revealed itsability to up- and downregulate a large number of human genes including those that are critical for neuronal development andmaintenance. We propose GHK tripeptide as a possible therapeutic agent against age-associated neurodegeneration and cognitivedecline.

1. Introduction

Today the most widespread neurological problems areconsidered neurodegenerative diseases of aging such asAlzheimer’s and Parkinson’s that rob people from theirgolden years causing early debilitation and dependencyon special care. Despite all advances in neuroscience inrecent decades, age-associated cognitive decline resultingfrom neurodegenerative processes in the brain remains achallenge for researchers and clinicians due to the highlycomplex nature of its pathogenesis.

Currently, the key processes leading to neurodegenera-tion are thought to be oxidative stress, disruption of transi-tional metal homeostasis, and neuroinflammation [1, 2].

There is also growing evidence that neurodegenerativediseases such as Alzheimer’s and Parkinson’s may be causedby detrimental environmental and dietary factors that alter

gene expression by means of DNA methylation and histonemodification [3].

Since the importance of oxidative stress in thedevelopment of age-related neurodegeneration is wellestablished, a number of antioxidant compounds areconsidered promising in prevention of neurodegenerativedisorders, including vitamin E, melatonin, green teapolyphenols, resveratrol, and others [4]. However, so far theclinical studies produce mixed results with many promisingapproaches such as vitamin E therapy failing to slow downthe progression of age-related neurodegenerative conditions[5]. There are also studies investigating an effect of metalchelators and dietary approaches aimed at reducing intakeof iron and copper [6]. Finally, researchers are investigatingdiverse compounds capable of favorably altering geneexpression (epigenetic modifiers), reversing effects ofenvironmental perturbagens [7].

2 Oxidative Medicine and Cellular Longevity

The human copper-binding tripeptide glycyl-L-histidyl-L-lysine (GHK) is a compound with a long history of safeuse in wound healing and antiaging skin care. Since itsdiscovery in 1973, almost four decades of extensive researchhave established its diverse beneficial actions in many organsand tissues including nervous tissue, skin, intestine, bone,and blood vessels. The molecule has a high affinity for Cu (II)and forms the chelate GHK-Cu. The GHK copper complex(or GHK-Cu) has been proven to exhibit antioxidant, anti-inflammatory, regenerative, and wound healing actions [8].Recent studies demonstrated that the GHK tripeptide up-and downregulates a large number of human genes, whichmay contribute to the pleiotropic health promoting effectsof its copper complex [9]. We propose that the GHK-Cu complex may act therapeutically against age-associatedneurodegeneration and cognitive decline.

2. Copper Binding Properties of the HumanTripeptide GHK

Human peptide GHK was isolated in 1973 as an activityin human plasma that caused old human liver tissue tosynthesize proteins like younger tissue [10]. In humanplasma GHK is present at about 200 micrograms/liter inmen of age 20–25 but declines to 80 micrograms/liter byage 60–80. Subsequent studies established this activity as atripeptide with an amino acid sequence glycyl-L-histidyl-L-lysine with a strong affinity for copper that readily formedthe complex GHK-Cu. Since GHK-Cu promotes cell growth,it was proposed that the GHK acts by delivering copperrequired for the cellular functions into the cell in a form thatis nontoxic and can be utilized by the cell [11].

The molecular structure of the GHK copper complex(GHK-Cu) has been extensively studied using X-ray crystal-lography, EPR spectroscopy, X-ray absorption spectroscopy,and NMR spectroscopy as well as other methods suchas titration. In the GHK-Cu complex, the Cu (II) ion iscoordinated by the nitrogen from the imidazole side chainof the histidine, another nitrogen from the alpha-aminogroup of glycine, and the deprotonated amide nitrogen ofthe glycine-histidine peptide bond (Figure 1). Since such astructure could not explain a high stability constant of theGHK-Cu complex (log10 = 16.44 versus 8.68 of the GHcopper complex, which is similar to the GHK-Cu structure),it was proposed that another amino group participatesin the complex formation. According to the recent studyby Hureau et al., the Cu (II) is also coordinated by theoxygen from the carboxyl group of the lysine from theneighboring complex. Another carboxyl group of lysinefrom a neighboring complex provides the apical oxygen,resulting in the square-planar pyramid configuration. Manyresearchers proposed that, at the physiological pH, GHK-Cu complexes can form binary and ternary structures whichmay involve amino acid histidine and/or the copper bindingregion of the albumin molecule. Lau and Sarkar foundalso that GHK can easily obtain copper 2+ bound to othermolecules such as the high affinity copper transport siteon plasma albumin (albumin binding constant log10 = 16.2

H

H

H

H

H H

H

H

H

H

O

O

OO

N

N

N

N N

N

Cu

Figure 1: Molecular structure of the tripeptide GHK-Cu. Insolution lysine carboxyl groups of neighboring complexes mayparticipate in a complex formation.

versus GHK binding constant 16 log10 = 16.44). It has beenestablished that copper (II) redox activity is silenced whencopper ions are complexed with the GHK tripeptide, whichallows the delivery of nontoxic copper into the cell [12–14].

3. The Copper Paradox

The brain is exceptionally rich in copper, which plays animportant role in its physiology and pathology. Copperdeficiency caused by bariatric surgery or gastrointestinalbleeding led to myelopathy (human swayback), paralysis,blindness, and behavioral and cognitive changes [15–17].Mice born and maintained on a copper-deficient diet had80% reduction in brain copper level at 6–8 weeks and hadneuronal and glial changes typical for neurodegenerativedisorders [18].

Despite unquestionable essentiality of copper, unreg-ulated copper ions may increase oxidative damage. It isknown that amyloid precursor protein (APP) implicated indevelopment of Alzheimer’s disease (AD) can convert Cu(II) into Cu (I) potentially increasing oxidative damage [19].However, it remains unclear whether copper accumulationin senile plaques of AD patients is a cause or a consequenceof pathological processes observed in AD. According toExley followed by Bolognin et al., only aluminum, but notcopper or iron, is capable of triggering amyloid precipitationand APP and tau181 protein overproduction [20, 21].The study by Kawahara et al. showed that copper andcarnosine attenuate neurotoxicity of another compoundinvolved in neurodegeneration—prion protein [22]. Bishopand Robinson observed that amyloid beta protein may beneuroprotective when combined with copper. According tothese authors, amyloid beta injected simultaneously withcopper was not toxic, while iron and zinc complexed withamyloid beta were more toxic than amyloid beta alone [23].

Since copper accumulates in senile plaques of ADpatients, some authors proposed dietary restriction of copperor intake of copper chelators as a preventive therapy forthe elderly. However, several studies demonstrated that AD

Oxidative Medicine and Cellular Longevity 3

patients have reduced, not elevated, brain and cerebrospinalfluid copper level [24, 25]. Currently, many authors suggestmild copper deficiency as a causative factor in AD andpossibly other neurodegenerative disorders [26, 27]. Ina placebo-controlled, double-blinded, randomized clinicaltrial, oral copper supplementation (8 mg/day) in 68 ADpatients had a beneficial effect on relevant AD biochemicalmarkers. The authors concluded that long-term intake ofcopper can be excluded as a causative factor in AD andmay in fact be protective [28]. In addition, several studiesrevealed molecular mechanisms underlying beneficial effectsof copper in AD, such as an inhibition of beta-amyloidpeptide production [29].

Due to high importance of copper in the brainmetabolism and a possible role of copper deficiency in thedevelopment of Alzheimer’s and other neurodegenerativedisorders, limiting of the dietary copper intake in elderly mayactually increase their chance of developing neurodegener-ative disorders. Another option is to use compounds thatcan form nontoxic complexes with Cu (II), preventing itsaccumulation in senile plaques and increasing its bioavail-ability. According to Narahara et al., β-citryl-L-glutamate—a compound that is abundant in the developing brain—has SOD-like activity when complexed with copper andmay be neuroprotective [30]. Rozga and Bal propose thathuman serum albumin (HSA) may be neuroprotective dueto its ability to bind both copper and amyloid beta protein[31]. Perrone at al. reported copper transfer from amyloidbeta to a copper binding domain of HSA that is similarto the tripeptide GHK’s structure with the same copper-binding histidine residue (DAHK) [32]. Thus GHK peptide,a natural copper-binding and copper-regulating moleculewith well-established regenerative and protective actionsin different organs and tissues, should be considered apromising therapeutic agent in preventing and correctingcopper imbalance in neurodegenerative disorders.

4. Antioxidant and Anti-InflammatoryProperties of GHK-Cu

The brain’s high metabolic activity results in elevated oxygenconsumption and constant production of reactive oxygenspecies (ROS) in mitochondria. At the same time, thebrain tissue is rich in unsaturated fatty acids and transitionmetal ions yet has relatively fewer antioxidants comparingto other organs creating favorable conditions for oxidativedamage. Since the blood-brain barrier prevents many dietaryantioxidants from entering the brain, it largely relies onendogenous antioxidants such as Cu- and Zn-dependentsuperoxide dismutase (Cu, Zn SOD1). This enzyme requiresmetal ions copper and zinc in order to be active. Hence,copper deficiency can lead to reduced SOD activity andincreased oxidative brain damage [33]. When pregnant ratswere kept on a copper-deficient diet, the embryos displayedlow SOD activity, increased super oxide anion radical level,and higher incidence of DNA damage and malformations[34]. Amyloid precursor protein (APP) that is implicated inAlzheimer’s disease (AD) development has copper binding

activity and can trap copper, rendering brain tissue copperdeficient. Transgenic mice overexpressing APP had reducedSOD1 activity in the brain. SOD1 activity was restored bycopper supplementation [35]. It has been shown that GHK-Cu increases the level of antioxidant enzymes and SODactivity, supposedly by supplying copper necessary for itsfunction [36].

GHK-Cu also reduces oxidative damage by modulatingiron levels. The presence of iron complexes in damagedtissues is detrimental to wound healing, due to the increasedlipid peroxidation in the presence of iron ions, as well asmicrobial infection mediated by iron. Pickart demonstratedthat GHK-Cu inhibited lipid peroxidation if the iron sourcewas ferritin. It was proposed that GHK-Cu binds to thechannels of ferritin involved in iron release and physicallyprevents the release of Fe (II). Thus, GHK-Cu exhibitsantioxidant function in wounds by inhibiting ferritin ironrelease in damaged tissues, preventing inflammation andmicrobial infections [37]. GHK-Cu produced a 75% reduc-tion of gastric mucosa homogenates of lipid peroxidationin the range 10–100 mM suggesting that copper-peptidecomplexes are able to effectively neutralize damaging oxygen-derived free radicals [38].

GHK (in this experiment the peptide alone, not itscopper complex, was used) has been proven to quenchalpha,beta-4-hydroxy-trans-2-nonenal—a toxic product offatty acids’ lipid peroxidation that play important role inthe pathogenesis of several age-related conditions includingAlzheimer’s disease, neuropathy, and retinopathy [39]. GHKpeptide was also able to quench acrolein—another toxicproduct of lipid peroxidation involved in the developmentof many age-related degenerative disorders. The authorsproposed that GHK may be used for prevention of some age-related pathologies, including Alzheimer’s disease [40].

Inflammation and oxidative damage due to the over-production of proinflammatory cytokines play an importantrole in the development of AD and other neurodegenerativeconditions [41]. In 2001 McCormack et al. establishedthat GHK-Cu decreased proinflammatory cytokine TGF-beta in human fibroblast culture [42]. In 2003 Canapp et al.demonstrated that GHK-Cu improved healing of ischemicwounds and suppresses inflammation by lowering the levelof acute-phase inflammatory cytokines such as TGF-beta andTNF-alpha [43].

5. GHK Stimulates Blood Vessel Growth

Vascular factors play an important role in the developmentof many neurodegenerative diseases of aging. Since the brainis a highly metabolically active organ and requires constantsupply of oxygen and nutrients, a well-developed, adequatevascular network is essential for its health [44].

From wound healing studies, it is known that GHK-Cuhelps reestablish blood flow into damaged tissues througha mixture of three actions: angiogenesis (new blood-vesselformation), anticoagulation, and vasodilation. GHK-Cuincreases the expression of basic fibroblast growth factorand vascular endothelial growth factor, both of which aidblood vessel formation [45]. In addition GHK-Cu’s ability

4 Oxidative Medicine and Cellular Longevity

to stimulate synthesis of collagen and elastin is useful inrestoring integrity of blood vessel walls.

Sage et al. observed that endothelial cells at the site ofan injury produce a protein called SPARC that containsGHK sequence. SPARC protein turned out to be abundantin all tissues that undergo rapid remodeling such as skinor embryonic tissues. When the tissue is damaged, tissueproteases break down SPARC, releasing an array of GHK andGHK-containing copper-binding peptides, which stimulatecell proliferation and new vessels growth. When blood flowis sufficiently restored, SPARC inhibits cell proliferation andgrowth, controlling new vessels progression [46].

6. GHK Increases Neurotrophins

Among compounds that have a protective effect and canreduce oxidative damage are some neurotrophic factors suchas brain-derived neurotrophic factor (BDNF) [47]. Thereis evidence that GHK increases production of neurotrophicfactors. Both Sensenbrenner et al. and Lindner et al. foundthat GHK stimulates the outgrowth of cultured nerves [48,49]. Ahmed et al. established that nerve stubs placed in acollagen tube impregnated with GHK (used without copper)had an increased production of nerve growth factor andthe neurotrophins NT-3 and NT-4 increased migration ofcells into collagen tube and sped up the regeneration ofnerve fibers. In addition, GHK also increased axon count andproliferation of the Schwann cells compared to the controlgroup [50].

7. GHK as a Gene Regulator

Epigenetic modification of gene expression is currentlyconsidered a link between the environment, aging, andneurodegeneration. It has been shown that some dietaryand environmental factors, such as certain toxins, may resultin abnormal DNA methylation and histone modification,altering gene activity. Recent studies revealed that somewell-known antioxidant and anti-inflammatory substancessuch as plant flavonoids may counteract these deleteriouschanges by modulating activity of certain genes, reducingglial inflammation and inhibiting production of neurotoxins[51, 52].

First evidence that GHK-Cu may regulate an activity ofcertain genes came from wound healing and skin remodelingstudies. By 1983, Pickart had established that GHK-Cu accel-erates wound healing and contraction, improves the takeof transplanted skin, and also possesses anti-inflammatoryactions [53, 54].

Subsequent studies directed by Maquart et al. (France)demonstrated that GHK-Cu at a very low, nontoxic con-centration (1–10 nanomolar) stimulated both the synthesisand breakdown of collagen and glycosaminoglycans [55, 56].It modulated an expression of both metalloproteinases andtheir inhibitors (TIMP-1 and TIMP-2), improving woundhealing and facilitating skin remodeling processes [57]. In2000 the same group demonstrated that GHK-Cu increasedmRNA for collagen, dermatan sulfate, chondroitin sulfate,and a small proteoglycan decorin [58].

In 2009, a group of researchers from the Seoul NationalUniversity (Republic of Korea) demonstrated that GHK-Cuin concentrations of 0.1–10 microM increases expressionof integrins and p63. Since these molecules are consideredproliferative markers of epidermal stem cells, the authorsconcluded that GHK-Cu helps to maintain an active prolif-erative state of epidermal stem cells [59].

Iorio et al. used a repository of transcriptional responsesto compounds, the Connectivity Map (cMap) [60], andMANTRA software (http://mantra.tigem.it/) to explore net-works of compounds producing similar transcriptionalresponses. GHK, as one of the compounds studied, increasedmRNA production in 268 genes while suppressing 167 [61].

Also, Hong et al. used genome-wide profiling to identifygenetic biomarkers (genetic signature) for metastasis-pronecolorectal cancer as well as their perturbagens—substancesthat modulated their expression. GHK suppressed RNA pro-duction in 70% of 54 human genes overexpressed in patientswith an aggressive metastatic form of colon cancer and wasactive at a low nontoxic 1 micromolar concentration [62].

8. The Connectivity Map andthe GHK-Affected Genes

Our own studies using the Broad Institute’s ConnectivityMap (cMap) showed that GHK activates numerous genesinvolved in nervous system physiology, development, andmaintenance. The Connectivity Map holds three GHK geneexpression profiles created using the GeneChip HT HumanGenome U133A Array. Two of the profiles emerged fromthe treatment of the PC3 cell line; the other profile camefrom the cell line MCF7. All cell lines were treated with GHKat 1 micromolar. By selecting all three gly-his-lys instanceswe determined which genes are affected. Most interestingare the genes that are remarkably up- and downregulated.Using the cMap one can enter amplitude thresholds forboth up- and downregulated genes in order to find thegenes whose expression are significantly altered. With an upgene amplitude threshold of 0.40 (equivalent to a 1.5-foldinduction) and a down gene amplitude threshold of −0.40(equivalent to a 1.5-fold repression), we discover that 76genes lay above the up threshold and 6 lay below the downthreshold.

In the cMap genes are represented by probe set IDsin the tag lists, which identify the up- and downregu-lated genes. Running the probe set IDs in the tag liststhrough the “Batch query” in the NetAffx Analysis Center(http://www.affymetrix.com/), annotations for each probeset are retrieved. Reviewing the annotations we observethat 5 genes associated with nerves are stimulated (mRNAexpression is increased) by GHK while none are suppressed(mRNA expression is decreased). These 5 genes are listed inTable 1 along with their corresponding average fold change.

9. GHK May Reverse Gene Silencing

Epigenetic silencing of certain genes is currently con-sidered the main reason for age-associated increase in

Oxidative Medicine and Cellular Longevity 5

Table 1

Probe set ID Gene symbolGene products and functions (from GENE database;http://www.ncbi.nlm.nih.gov/gene)

Fold change∗

214484 s at SIGMAR1

Sigma nonopioid intracellular receptor 1, plays an important role in thecellular functions of various tissues associated with the endocrine,immune, and nervous systems. Mutations are implicated in early-onsetdementia and neurodegeneration.

2.46

205231 s at EPM2ALaforin, a dual-specificity phosphatase associates with polyribosomes.This gene is defective in a neurodegenerative disorder associated withepileptic seizures (Lafora disease). Possibly a repair enzyme.

2.32

204860 s at NAIPApoptosis inhibitory protein; functions include suppression of neuronalapoptosis

2.13

208229 at FGFR2Fibroblast growth factor receptor 2, influences mitogenesis anddifferentiation, important in embryonic brain development.

1.9

209897 s at SLIT2Slit homolog 2, neuronal repellent factor, nervous system development;downregulation of this gene by neuronal differentiation factor promotestumor growth in neuroblastomas [63].

1.67

∗This represents the average fold change of gene expression of the three GHK instances profiled in the cMap. Furthermore, it should be noted that all instances

were performed using doses of GHK at 1 micromolar. Peak cellular responses to GHK have been recorded at 1 nanomolar; higher doses can reduce the cellularresponse [56].

tumorogenesis, oxidative stress, and inflammation. It iscommonly accepted that human health is at its best untilapproximately the age of 20–25. It then begins to decline inlater years. Recent genetic studies demonstrated accumula-tion of altered gene products in various tissues starting fromthe age of 20–25 [64]. In later decades of life, the genesproduce less regenerative proteins but more inflammatoryand oncogenic genes. The key enzymes implicated in genesilencing are the family of histone deacetylase proteins(HDACs). Inhibitors of selected HDACs possess neuropro-tective and neuroregenerative properties in animal modelsof brain diseases and have been suggested as promisingtherapeutic drugs [65]. Results from the Broad Institute’sConnectivity Map and ChemBank found that GHK is astrong inhibitor of several HDACs. Although the data wascollected for GHK without copper, we cannot exclude thepossibility that the actual gene regulator was the GHKcopper complex formed in the culture media, since, as itwas mentioned above, GHK can easily obtain copper fromother biological molecules such as albumin. It is also possiblethat GHK and GHK-Cu have complimentary effects on geneactivity. At present, it is not always possible to track geneeffects to its protein product; however, we may conclude fromall experimental data on GHK-Cu effects that the reversing ofgene silencing by GHK has protective and health-promotingbenefits.

10. Therapeutic Administration of GHK-Cu

It is possible that administration of GHK-Cu could be usedas a preventive and regenerative therapy for senescent ordamaged brain tissue. Using GHK-Cu has an advantage overusing just GHK, since it alleviates copper deficiency withoutthe risk of oxidative damage. Even though it is yet not clearwhether or not the GHK-Cu peptide can pass the blood-brain barrier, there is a high possibility that it will do so,since GHK-Cu has a very high uptake into human skin,

easily passing through the lipids of the epidermal barrier[66, 67]. The peptide could be administered intravenouslyor orally when encapsulated into liposomes. Strong systemicwound healing was induced in pigs at about 1.1 mg GHK-Cu per kilogram body weight which would correspond toabout 75 mgs in humans. This is about 300-fold below GHK-Cu’s toxic action (lowering of blood pressure). Much lowerdosages may also be effective since GHK-Cu’s actions on cellsgenerally occur at a 1 nanomolar concentration [68].

11. Conclusion

The multifaceted nature of age-associated cognitive declinecalls for complex approaches that address all key factorsinvolved in the development of neurodegenerative disor-ders such as oxidative stress, neuroinflammation, disruptedbioavailability of copper, impaired circulation, and alteredgene expression.

The human tripeptide GHK has a long history of safeuse in wound healing and skin care; it is naturally occurring,nontoxic, and is active at a very low nanomolar concentra-tion. It readily forms complexes with copper, regulating itsmetabolism and improving its bioavailability. It possessesantioxidant, anti-inflammatory, and regenerative properties,improves circulation, supports stem cell functions, andpromotes nerve outgrown and synthesis of neurotrophicfactors. Recent studies demonstrated its ability to regulate alarge number of human genes. At 1 micromolar it was ableto suppress 70% of genes overexpressed in metastatic coloncancer. It upregulates p63 and integrins in epidermal stemcells, increases collagen, glycosaminoglycans, and decorinexpression. Our studies with the Broad Institute’s Connec-tivity Map revealed its ability to regulate a large number ofhuman genes including those that are involved in nervoussystem physiology, development, and maintenance.

Even though it is not always possible to distinguishbetween activity of GHK peptide and its copper complex

6 Oxidative Medicine and Cellular Longevity

GHK-Cu, we strongly believe that for the future therapeuticalapplications GHK-Cu should be used. Numerous studiesdemonstrating pleiotropic health promoting and antiageactivity of the GHK-Cu peptide together with recent studiesrevealing gene regulating activity of GHK suggest that thiscompound may belong to a class of epigenetic modi-fiers capable of exhibiting broad protective and restorativeactions, reducing harmful epigenetic changes caused byenvironmental perturbagens. The GHK-Cu peptide shouldbe considered a promising neuroprotective agent capableof preventing the development of common age-associatedneurodegenerative disorders.

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